Denitrification of solutions is useful for many reasons, such as limiting the total nitrogen discharged in wastewater to comply with local permits. Other reasons include: improving freshwater quality; controlling alkalinity and oxygen recovery, producing stabilized effluent, and reducing issues stemming from sludge accumulation in the clarifier.
Removing nitrogen from wastewater requires understanding the different forms of nitrogen and some commonly referred to terms:
Total Nitrogen (TN) is the sum of all nitrogen forms or:Total Nitrogen=TKN+NO2—+NO3—where:                TKN stands for Total Kjeldahl Nitrogen, which is the sum of: NH3+Organic Nitrogen;        
NH3 stands for Ammonia Nitrogen or Ammonium ion (NH4—);
Organic Nitrogen is derived from amino acids, proteins, urea, uric acid, etc.;
NO2— represents a Nitrite ion;
NO3— represents a Nitrate ion; and
N2 represents Nitrogen Gas.
Refractory Nitrogen cannot be decomposed biologically.
Alkalinity is defined as the ability to resist a drop in pH. For every part ammonia (NH3) converted to nitrate (NO3—), 7.1 parts of alkalinity are depleted, and for every part nitrate (NO3—) removed, 3.6 parts of alkalinity are recovered.
An anoxic zone is a basin, or portion that is mixed, but not aerated. The dissolved oxygen levels must be less than 1.0 mg/L, and avoid as low as 0.0 mg/L. In an anoxic zone, denitrifying bacteria derive oxygen from the nitrate (NO3—) compounds.
Nitrification and denitrification are two terms that are commonly misunderstood. Both are individually distinct processes. Nitrification is the conversion of ammonia (NH3) to nitrate (NO3—). This is a two-step process involving oxygen and two types of bacteria, Nitrosomonas and Nitrobacter, known collectively as nitrifiers, represented as follows:Ammonia (NH3)+Oxygen (O2)+Alkalinity+Nitrosomonas=Nitrite (NO2—)+Oxygen (O2)+Alkalinity+Nitrobacter=Nitrate (NO3—)Nitrite (NO2—) is unstable and is easily converted into nitrate. The total conversion of ammonia (NH3) to nitrate (NO3—) requires 4.6 parts oxygen and 7.1 parts alkalinity to convert 1 part ammonia (NH3).
Denitrification is the conversion of nitrate (NO3—) to nitrogen gas (N2). Heterotrophic bacteria use nitrate (NO3—) as an oxygen source under anoxic conditions to break down organic substances as follows:Nitrates (NO3—)+Organics+Heterotrophic bacteria=Nitrogen Gas+Oxygen+Alkalinity
In practice, only certain forms of nitrogen are monitored in wastewater treatment facilities with specialized testing equipment. Testing for TKN involves a test that many wastewater treatment facility laboratories are not equipped to perform. If testing for TKN is not possible, other methods are used for monitoring the nitrogen cycle.
Typically, ammonia (NH3) values are approximately 60% of the TKN values, and the organic nitrogen generally is removed to the settled sludge. Also, total Kjeldahl nitrogen (TKN) generally equals 15-20% of the Biochemical Oxygen Demand (BOD) of the raw sewage. Testing the following aid in monitoring and controlling the nitrogen cycle: pH, alkalinity, ammonia (NH3), nitrite (NO2—) and nitrate (NO3—). All major laboratory supply companies sell field test kits that are inexpensive, easy to use, and provide quick relatively accurate results.
Having a good understanding of the form and extent of nitrogen in a wastewater treatment facility requires a good sampling program that gives a complete profile of the system. The first sampling point should test the raw influent, or primary effluent if the system has a primary clarifier. Typically, what enters the system is high in alkalinity and ammonia (NH3) with little to no nitrite (NO2—) or nitrate (NO3—). A quick way to determine if additional alkalinity may be needed is to multiply the amount of ammonia (NH3) by 7.1 mg/L. If this number exceeds the influent alkalinity concentration, sodium hydroxide or lime may be needed to be added to the aeration tank.
pH is significant because, when ammonia (NH3) begins converting to nitrate (NO3—) in the aeration tank, many hydrogen ions are released. When alkalinity drops below 50 mg/L, pH can drop dramatically. The pH of the aeration tank should never drop below 6.5, otherwise desired biological activity will be inhibited and toxic ammonia (NH3) can bleed through the system to the environment.
Ammonia (NH3) should have extremely low concentrations. Nitrite (NO2—) should be very low to non-detectable, with the majority of the nitrogen in the nitrate (NO3—) form. If a suitable environment is maintained in the aeration tank, most of the ammonia (NH3) will be converted to nitrate (NO3—) by the time it leaves the tank.
All tested nitrite (NO2—) levels should be very low. High levels of nitrite (NO2—) in the system indicate an existing or anticipate problem with the nitrification cycle.
Nitrosomonas bacteria are hardier than Nitrobacter bacteria. If the Nitrobacter bacteria die off, the Nitrosomonas bacteria will continue working on the ammonia (NH3) and the cycle will overload with high levels of nitrite (NO2—). An effluent with high nitrite (NO2—) concentrations is difficult to disinfect because of the tremendous chlorine demand it poses.
Other problems also can occur during nitrification. A decrease in the aeration tank pH due to insufficient alkalinity causes ammonia (NH3) to bleed through the system, which causes decreased microbiological activity. Other factors that prevent complete nitrification include: a lack of dissolved oxygen; high mixed liquor suspended solids; low mean cell retention time; and cold temperatures.
All of these factors can inhibit the nitrification cycle. High ammonia (NH3) discharges can affect toxicity testing. High nitrite (NO2—) levels will cause a tremendous chlorine demand making disinfection difficult, jeopardizing fecal coliform limits. Leaving sludge that is high in nitrate (NO3—) too long in a secondary clarifier can cause it to rise to the surface when the nitrogen gas is released. This is messy and jeopardizes TSS limits.
Although problematic, nitrifying wastewater is important for many reasons. Aside from permit limits, ammonia (NH3) is toxic to fish and other aquatic life. Ammonia (NH3) discharges also place a very high oxygen demand on the receiving streams. Nitrification also aids in producing a highly stabilized effluent.
When all of the ammonia (NH3) is converted to nitrate (NO3—), it is removed from the system or denitrified. Denitrification requires an anoxic zone within the wastewater treatment facility. Regardless of where and how it is done, the principles of operating an anoxic zone are always the same. First, dissolved oxygen levels must be as low as possible without reaching 0.0 mg/L. A safe target point to avoid septicity while starting an anoxic zone is 0.5 mg/L. A good operating point is 0.2 mg/L.
Second, a carbon source must exist for denitrification to occur. A “carbon source” supplies life energy to the bacteria. A carbon source compound may include additional elements to carbon, such as hydrogen and oxygen. The bacteria also must have oxygen to be able to utilize the carbon. They obtain oxygen from the easiest sources in the order of: (1) free and dissolved oxygen; (2) nitrate (NO3—); and then (3) sulfate (SO4—). If the environment has no free or dissolved oxygen, the bacteria obtain oxygen by breaking down nitrate (NO313 ) returned to the anoxic zone in the form of activated sludge. As the bacteria use the nitrate (NO3—) as an oxygen source to break down the carbon, their food source, nitrogen gas is released to the atmosphere as follows:bacteria+Carbon Source+Nitrate (NO3—)=Nitrogen Gas (N2)+Carbon Dioxide (CO2)+3.6 parts Alkalinity+Water (H2O)When all of the nitrate (NO3—) is used up, the bacteria look for oxygen from available sulfate (SO4—). As the sulfates are used up, the free sulfides will combine with hydrogen to form hydrogen sulfide, which has a characteristic “rotten egg” odor. Thus, treatment plant operators are can always tell when all of the nitrate (NO3—) is being converted into nitrogen gas (N2).
Raw influent can be used as a carbon source. However, most treatment plants supplement the carbon source, for example, by injecting methanol, ethanol or other like carbon sources. Roughly 2.0-2.5 parts methanol is required for every part nitrate (NO3—) that is denitrified.
The mixed liquor suspended solids concentration must be kept in balance with the carbon source supply. In other words, the carbon source-to-microorganisms ratio should be in the proper range, on the lower end, for the type of process operating. The pH of the anoxic zone should be close to neutral (7.0) and never drop below 6.5.
Optimal denitrification occurs when as much as possible of the nitrate (NO3—) is converted into nitrogen gas (N2). Achieving this requires a sufficient amount of a carbon source so that the indigenous heterotrophic bacteria will consume all of the dissolved oxygen as well as the oxygen from the nitrate (NO3—), thereby converting as much as possible of the nitrate (NO3—) into nitrogen gas (N2).
Many carbon sources for denitrification have been studied and utilized in wastewater treatment systems. The most popular include the simple alcohols methanol [15] and ethanol [3]. Acetate in the form of either acetic acid [1] or some acetate salt, e.g. sodium acetate [7], has also been used. “Acetate” refers to either the ion, as in sodium acetate, or the substituent group, as in ethyl acetate [6]. The studies frequently indicate acetate [7] as the most effective of these listed, and the many other compounds subjected to these studies.

However, these compounds leave much to be desired for use as denitrification carbon sources for wastewater treatment units, especially on-site wastewater treatment units. Acetic acid is a solid and corrosive in the pure state. When diluted to safer levels, it becomes very bulky. Acetate salts also are hazardous solids, and face the same fate on adequate dilution. Since acetate salts of sodium or potassium are solids, they must be dissolved for pumping by metering devices. These solutions are bulky, and leave solid residue on drying that can foul the equipment. The residual from utilization by the bacteria is an increase in alkalinity that is impractical to control in an unattended system.
Among the other compounds used for larger plants are simple alcohols, like ethanol [3] and methanol [15], depicted above, and polyalcohols like glycerol [2]. These alcohols also have their own limitations with respect to on-site use.

Fatty acids, monoglycerides, and diglycerides derived from the saponification of fats also can be used as carbon sources. Short-chain fatty acids are water soluble, while longer-chain fatty acids reduce solubility so that they become surfactants, with soap being the classic example. Their esters are insoluble.
Fats and oils are esters of glycerin and 3 long chain fatty acids, and are also known as triglycerides [8]. Fatty acids that have carbon-to-carbon double bonds are referred to as “unsaturated fatty acids” [5].

These traditional supplementary carbon sources, methanol and ethanol, have undesirable characteristics, especially for on-site use, including acute toxicity; volatile; flammable; and form explosive vapor mixtures with air in confined spaces. Ethanol, while grain derived in its natural form is highly regulated and expensive. Cheaper, unregulated denatured ethanol, in excess amounts, inhibits decomposition. It also, when decomposed, yields byproducts including benzene, ethylene, toluene, and xylene, which should not be released into the environment. Since an excess of carbon source is needed to ensure that a sufficient amount of heterotrophic bacteria will locate and convert as much as possible of the nitrate (NO3—) into nitrogen gas (N2), using denatured ethanol causes less and less conversion and could build up in the treatment tank and stifles decomposition. Although ethanol is a good carbon source, it must be converted to acetaldehyde [14], and then acetate before the bacteria can utilize it.

What is needed is a carbon source compound that can deliver the effectiveness of acetate with none of the above-mentioned issues, and has only residuals that can be assimilated by the denitrifying bacteria.
One such compound class could be the acetate esters of glycerol. Other polyalcohols, such as ethylene glycol [16], propylene glycol [17] and butylene glycol [19]-[22] also might serve as carriers of acetate in the form of esters, which are combinations of alcohols and organic acids. One example might be 1,2-propylene glycol diacetate [18]. Ethanol and acetic acid combine to form ethyl acetate [6], depicted above.

Many wastewater treatment facilities perform single-tank denitrification by creating and utilizing anoxic zones. Some examples are:                (1) Constructing a dedicated anoxic zone at the head of the aeration tank by installing a baffle and mechanical mixers;        (2) Utilizing the first ¼ to ⅓ of the aeration basin as an anoxic zone by throttling the aeration system diffusers valves to allow mixing without transferring dissolved oxygen. A dissolved oxygen probe in the aeration tank tied into a variable frequency drive that sends a signal to the blowers, providing a continuous dissolved oxygen level as determined by the set points; and        (3) Utilizing timers to cycle the aeration system on and off which allows the whole aeration basin to be used intermittently as an anoxic zone.These approaches do not completely denitrify the wastewater so treated.        
What are needed, and not taught or suggested in the art, are an apparatus for and method of denitrifying a solution that employs an inexpensive, non-toxic, unregulated carbon source for heterotrophic bacteria to reduce all nitrate (NO3—) in solution.